Plant Science 156 2000 159 – 167
Histological analysis of indirect somatic embryogenesis in the Marsh clubmoss Lycopodiella inundata L. Holub Pteridophytes
N. Atmane
a
, A.S. Blervacq
a,
, N. Michaux-Ferriere
b
, J. Vasseur
a
a
Laboratoire de Physiologie Cellulaire et Morphogene`se Ve´ge´tales, USTL, Baˆtiment SN
2
, F-
59655
Villeneu6e d
’
Ascq Ce´dex, France
b
Laboratoire d
’
histologie, CIRAD-BIOTROP, BP
5035
, F-
34032
Montpellier Ce´dex, France Received 6 October 1999; received in revised form 1 March 2000; accepted 1 March 2000
Abstract
An efficient in vitro plant regeneration method was developed for Lycopodiella inundata L. Holub, an endangered medicinal Lycopod Pteridophytes. Vegetative apices were used as explant material. Nodular calluses were established after three cycles 13
weeks each on a medium containing a few minerals and organic compounds and supplemented with 0.05 mM 3-indolebutyric acid IBA and 1.4 mM kinetin Kin. Propagation was achieved every 13 weeks on this callus medium CM. When nodular calluses
were transferred on a medium supplemented with 2.5 mM IBA and 0.33 mM gibberellic acid GA
3
designated as embryogenic medium EM, organized structures appeared and developed into plantlets. Development phases were characterized by histological
studies. Some phases of zygotic embryogenesis previously described for Lycopods were observed in L. inundata. Histological analyses established that an indirect somatic embryo was derived from a single embryogenic cell by following the zygotic
developmental pathway. As this phenomenon has not previously been reported in Lycopods, a comparison between somatic and zygotic embryos is discussed based upon morphology and histology. © 2000 Published by Elsevier Science Ireland Ltd. All rights
reserved.
Keywords
:
Callogenesis; Histology; Indirect somatic embryogenesis; Lycopodiella inundata; Pteridophytes www.elsevier.comlocateplantsci
1. Introduction
Marsh clubmoss, Lycopodiella inundata L. Holub is commonly found in hygrophile and olig-
otrophic environments. It is an ancestral vascular plant that belongs to the Lycopodiales order
which also includes Huperzia selago and Ly- copodium cernuum [1]. Non-regulated collecting of
these plants for industrial or pharmaceutical uses endangers their existence [1,2]. The wild popula-
tion is a source of spores for condoms [3,4], and is traditionally used in practical medicine [5] and
homeopathy [6]. New opportunities have been opened since alkaloids from Lycopodiales were
tested for the alleviation of symptoms related to Alzheimer’s dementia [7] and as a drug against
nerve agent toxicity [8]. In vitro propagation is currently initiated on
ferns Pteridophytes from sporophyte fragments such as creeping stems. That process usually pro-
duces a typical nodular callus. Meristematic nod- ule propagation is also reported as an efficient
method for in vitro plant regeneration of Matteuc- cia struthiopteris [9]. While ferns are often propa-
gated by in vitro methods, no propagation via axenic spore germination or calluses derived from
sporophyte has been described for Lycopods, in- cluding L. inundata.
According to its recent protected status as an endangered plant species, and because of the phar-
maceutical importance of its alkaloids, it is impor- tant to micropropagate L. inundata. Axenic spore
germination was not obtained, so fragments of the sporophyte were chosen as the explant. Vegetative
Abbre6iations
:
CM, callus medium; EM, embryogenic medium; GA
3
, gibberellic acid; IBA, 3-indolebutyric acid; Kin, kinetin; MS, Murashige and Skoog medium; SE, somatic embryogenesis.
Corresponding author. Tel.: + 33-3-20434016; fax: + 33-3- 20336044.
E-mail address
:
blervacquniv-lille1.fr A.S. Blervacq. 0168-945200 - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 2 4 4 - 2
apices of L. inundata were responsive. The proto- col for propagation [10] was optimized. Organo-
genesis was studied using cytological techniques and concluded to result from the direct shooting
from axillary meristems [10].
In this work, we report on the establishment of nodular callogenesis for propagation. The histo-
logical analysis of the initiation and development of somatic embryos is detailed and compared with
zygotic embryogenesis in Lycopods. Somatic em- bryogenesis SE among Plantae Pteridophytes-
Spermaphytes is discussed.
2. Materials and methods
